Protein kinases play a key role in cell growth and differentiation. The p21-activated proteins kinases (PAKs) are related to a yeast serine/threonine protein kinase, Ste20. Ste20 is a member of a growing family of regulatory enzymes that may play roles in diverse phenomena such as cellular morphogenesis, the stress response and the pathogenesis of AIDS.
The growth, differentiation, maintenance and senescence of cells requires the transmission of signals. These signals can be derived from extracellular stimuli, such as hormone interaction with its receptor or physiological stress, or may be derived intracellularly from developmental programs.
The signals are transmitted via a signal cascade wherein proteins are phosphorylated or dephosphorylated in sequence. Protein kinases, the enzymes that phosphorylate, play a key role in many signaling pathways and therefore protein kinases play a key role in cell growth and differentiation. Protein kinases fall into two broad categories, tyrosine kinases (those that add phosphate to tyrosine residues) and serine/threonine kinases (those that add phosphate to either serine or threonine residues).
One group of signaling pathways involving protein kinases are the mitogen-activated protein (MAP) kinase cascades. The MAP kinase cascades are now recognized to participate in diverse signal transduction pathways rather than only mediating mitogenic signals from all surface receptors (Davis, R. J., 1994, Trends in Biochem Science, 19:470-473).
Genetic analyses in yeast have made it possible to identify some of the components of MAPK pathways, and to clarify the diversity of their functions and regulation (Herskowitz, I., 1995, Cell 80:187-197). Mammalian cells have at least three MAPK pathway subtypes, the ERK (extracellular signal-regulated protein kinase) pathway, JNK (c-Jun NH2-terminal kinase)/SAPK (stress-activated protein kinase) pathway, and P38/Mpk2 pathway; together, these pathways mediate a wide variety of physiological responses (Davis, R. J., 1994, Trends in Biochem. Science 19:470-473; Derijard, B. et al., 1994 Cell 76:1025-1037; Kyriakis, J. M. et al., 1994 Nature 369:156-160; Han, J. et al., 1994 Genes Dev. 3:1336-1348; Lee, J. C. et al., 1994 Nature 372:739-746; Rouse, J. et al., 1994 Cell 78:1027-1037). MAPKs are activated by sequential protein phosphorylation reactions. The basic framework of the MAP kinase pathway, where MAPK is phosphorylated on Thr and Tyr residues and activated by MAPK kinase (MAPKK), before which MAPKK is itself phosphorylated and activated by MAPKK kinase (MAPKKK), is common from yeast to mammals (Nishida, E. and Gotoh, Y., 1993 Trends in Biochem. Science 18:128-131; Davis, 1994; Herskowitz, 1995; Marshall, C. J., 1994 Curr. Opin. Genet. Dev. 4:82-89).
A new kinase group activated by G-protein and thought to act as MAPKKK kinase (MAPKKKK) has been identified in both yeast and mammals. In budding yeast, this new kinase group is known by its prototype member Sterile 20 (Ste20). Ste20 is activated by the xcex2xcex1 complex released from the heterotrimeric G protein complex upon pheromone receptor stimulation, and in turn activates Ste11 (a MAPKKK) (Leberer, E. et al., 1992 EMBO J. 11:4815-4824; Ramer, S. W. and Davis, R. W., 1993 Proc. Natl. Acad. Sci. U.S.A. 90:452-456). In mammals, this new kinase group is known by its prototype member p21-activated protein kinase (PAK). PAK (now called xcex1-PAK) has been identified as a protein kinase activated by the Rho family of small G-proteins, Rac1 and Cdc42 (Manser, E. et al., 1994 Nature 367:40-46); PAK also shows sequence similarity to yeast Ste20. Recently it has been clarified that PAK comprises a protein kinase family composed of several PAK isoforms, hPAK65 (Martin, G. A. et al., 1995 EMBO J. 14:1970-1978), MPAK-3 (Bagrodia, S. et al., 1995 J. Biol. Chem. 270:22731-22737 and xcex2-PAK (Manser, E. et al., 1995 J. Biol. Chem. 270:25070-25078), all of which are able to interact with Cdc42 and Rac1. Rac1 and Cdc42 have been implicated not only in cell motility (Ridley, A. J. et al., 1992 Cell 70:401-410; Kozma, R. et al., 1995 Mol. Cell. Biol. 15:1842-1952; Nobes, C. D. and Hall, A., 1995 Cell 81:53-62), but also in the preferential activation of the JNK/SAPK and p38/Mpk2 pathways rather than the ERK pathway (Coso, O. A. et al., 1995 Cell 81:1137-1146; Minden, A. et al., 1995 Cell 81:1147-1157; Olson, M. F. et al., 1995 Science 269:1270-1272). This is in contrast to another small G-protein, Ras, which predominantly activates the ERK pathway through Raf activation (Minden, A. et al., 1994 Science 266:1719-1723). Although a direct interaction between PAKs and components of the JNK/SAPK and p38/Mpk2 pathways has not yet been demonstrated, these observations raise the intriguing possibility that PAK or PAK-related proteins mediate the signals from Rac1 and Cdc42 to the JNK/SAPK and p38/Mpk2 pathways, and, furthermore, that G-proteins differentially regulate MAPK pathways to achieve various physiological responses.
In contrast to the above-mentioned kinases, a group of Ste20-related kinases that lack the putative Cdc42/Rac1-binding domain has been identified in both yeast and mammals: Sps1, an upstream regulator of the MAPK pathways (Freisen et al., Genes and Dev., 9:2162-2175 (1994) and MST-1 in Mammals; Creasy, C. L. and Chernoff, J., 1995 J. Biol. Chem. 270:21695-21700). Although the specific activation of the SAPK pathway by GCK has recently been reported (Pombo, C. M. et al., 1995 Nature 377:750754), the upstream and downstream signaling pathways of this group of kinases remain to be clarified. Sterile 20-related kinases are regulatory molecules involved in mitogenic signaling as well as other cellular phenomena such as morphology and motility. These phenomena are important factors in development, cell differentiation, cancer and metastases. Therefore, the polynucleotides and polypeptides of the present invention allow manipulation of the signaling pathways involved and will allow the development of reagents to modulate the signaling pathways involved in these important cellular phenomena.
The present invention relates to isolated polynucleotides encoding novel members of the Ste20 family of serine/threonine protein kinases, and the recombinantly produced polypeptides encoded by said polynucleotides.
The present invention is drawn to a purified nucleic acid comprising at least 45 continuous nucleotides of a nucleic acid sequence provided in SEQ ID NO: 3. The purified nucleic acid can comprise SEQ ID NO: 3, a complementary sequence or a sequence having greater about 600 bases in length, wherein said sequence hybridizes to SEQ ID NO: 3 under stringent conditions.
The present invention is also drawn to a purified nucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ:ID NO: 9, a sequence complementary to either SEQ ID NO: 1 or 9 and a sequence greater than 500 bases in length wherein said sequence hybridizes to SEQ ID NO: 1 or 9 under stringent conditions.
The present invention also encompasses expression vectors comprising the polynucleotides of the present invention and host cells harboring said vectors.
The present invention is also drawn to a purified nucleic acids encoding an amino acid sequence comprising SEQ ID NOs.: 2, 4, or 10. In one embodiment, the purified nucleic acid sequence comprising a nucleic acid sequence encoding at least 100 continuous amino acids of an amino acid sequence provided in SEQ ID NO: 2 or SEQ ID NO: 10. In another embodiment, the purified nucleic acid sequence comprising a nucleic acid sequence encoding at least 54 continuous amino acids of an amino acid sequence provided in SEQ ID NO: 4. The method of the present invention is also drawn to a method of making polypeptides encoded by SEQ ID NOs.:1, 3, or 9 comprising, transfecting a host cell with an expression vector comprising SEQ ID NOs.: 1, 3, or 9 and isolating the expressed protein.
The present invention is further drawn to polypeptides comprising SEQ ID NOs.: 2 or 10 and biologically active fragments thereof, polypeptides functionally equivalent to polypeptides comprising SEQ ID NOs.: 2 or 10 or fragments thereof, antibodies that bind to polypeptides encoded by SEQ ID NOs.:1, 3, or 9.
The present invention is further drawn to a serine/threonine kinase comprising the amino acid sequence of SEQ ID NOs. 2, 4, or 10.
The protein kinases described herein have homology with a family of proteins that appear to function in the mitogen-activated protein (MAP) kinase cascade. The present invention also relates to methods of using the polynucleotides and polypeptides described herein to detect, isolate and characterize elements upstream and downstream of the novel kinase in the signal transduction pathway using assays well known in the art such as kinase assays or co-immunoprecipitation assays, or combinations thereof. Furthermore, polypeptides of the present invention include biologically active fragments of the proteins described herein. Such proteins and biologically active fragments are useful to generate antibodies that specifically bind the proteins of the present invention. The biologically active fragments are also useful as tools to study the activity of the protein. Altered forms of the polypeptides are within the scope of the present invention and can be used to study the activity of downstream elements in the signaling pathway or to generate specific antibodies.
The isolated polynucleotides and polypeptides of the present invention provide the advantage of being able to conveniently manipulate the genes, gene products and expression level of the gene product, to facilitate understanding of how Ste20 and Ste20 family members regulate signal transduction in the cell.